PROJECT SUMMARY Reversible serine/threonine phosphorylation of proteins plays an essential regulatory function in numerous cellular processes. Type 2C protein phosphatases (PP2Cs) represent a major class of Ser/Thr phosphatases, and defects in several human PP2Cs have been implicated in cancer, diabetes, heart disease, neural disorders, and stress signaling. However, little is known about the mechanisms by which PP2C activity is regulated. The plant hormone auxin regulates virtually every aspect of plant growth and development. Small Auxin Up-RNA (SAUR) genes represent the largest class of auxin-induced genes. The SAUR19-24 subset of highly related SAUR proteins specifically interact with and inhibit the enzymatic activity of PP2C.D family phosphatases to promote cell expansion. In part, this involves SAUR proteins preventing PP2C.D-mediated dephosphorylation of a key regulatory site of plasma membrane H+-ATPases. The long-term goal of this project is to thoroughly understand the molecular mechanisms underlying auxin-mediated control of plant growth and development. More specifically, the work outlined in this proposal will characterize and illuminate the mechanism by which SAUR proteins regulate PP2C.D phosphatases to control auxin-mediated cell expansion and other aspects of growth and development. The proposed studies include genetic, molecular, biochemical, and structural approaches to elucidate the regulatory mechanisms by which SAURs control PP2C activity in the model plant Arabidopsis thaliana. This plant provides a powerful genetic system for investigating conserved regulatory processes within multicellular eukaryotes. PP2C.D functions will be revealed through both gain- and loss-of-function genetic analyses of saur and pp2c.d mutant and transgenic lines, and the regulatory interactions between these two protein families elucidated. Secondly, phosphoproteomic profiling experiments will be conducted to biochemically define PP2C.D regulated pathways and identify potential phosphoprotein substrates important for auxin-mediated growth. Lastly, the structure of a SAUR-PP2C.D complex will be determined and tested in biochemical and genetic assays to illuminate the molecular mechanism of SAUR inhibition of PP2C.D activity at atomic resolution. The findings from the proposed experiments will likely have direct parallels to the mechanisms human cells employ to regulate PP2C activity, as PP2C structure and function are both highly conserved. Such detailed understanding of PP2C regulatory mechanisms will facilitate the development of novel therapeutic strategies to alter PP2C activity and combat disease. Further, as humans depend on plants for sources of food, fiber, medicine and fuel, the proposed studies will elucidate plant growth control by the SAUR-PP2C.D regulatory module and potentially lead to novel strategies for manipulating plant growth to benefit human health.